Part1:Acteoside Suppresses RANKL-Mediated Osteoclastogenesis By Inhibiting C-Fos Induction And NF- KB Pathway And Attenuating ROS Production
Mar 05, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Seung-Youp Lee1,2., Keun-Soo Lee3.¤, Sea Hyun Yi2., Sung-Ho Kook, Jeong-Chae Lee22,3*
Abstract
Numerous studies have reported that inflammatory cytokines are important mediators for osteoclastogenesis, thereby causing excessive bone resorption and osteoporosis. Acteoside from cistanche herb, the main active compound of Rehmannia glutinosa, which is used widely in traditional Oriental medicine, has anti-inflammatory and antioxidant potentials. In this study, we found that acteoside markedly inhibited osteoclast differentiation and formation from bone marrow macrophages (BMMs) and RAW264.7 macrophages stimulated by the receptor activator of nuclear factor-kappaB (NF-kB) ligand (RANKL). Acteoside pretreatment also prevented bone resorption by mature osteoclasts in a dose-dependent manner. Acteoside (10 mM) attenuated RANKL-stimulated activation of p38 kinase, extracellular signal-regulated kinases, and c-Jun N-terminal kinase, and also suppressed NF-kB activation by inhibiting phosphorylation of the p65 subunit and the inhibitor kBa. In addition, RANKL-mediated increases in the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and in the production of tumor necrosis factor-a, interleukin (IL)-1b, and IL-6 were apparently inhibited by acteoside pretreatment. Further, oral acteoside reduced ovariectomy-induced bone loss and inflammatory cytokine production to control levels. Our data suggest that acteoside inhibits osteoclast differentiation and maturation from osteoclastic precursors by suppressing RANKL-induced activation of mitogen-activated protein kinases and transcription factors such as NF-kB, c-Fos, and NFATc1. Collectively, these results suggest that acteoside may act as an anti-resorptive agent to reduce bone loss by blocking osteoclast activation.
Introduction
Bone is constantly remodeled by balanced osteoclast and osteoblast activity [1]. Osteoclasts arise from hematopoietic precursor cells of the monocyte/macrophage lineage, while osteoblasts are of the mesenchymal lineage [2]. Abnormal osteoclast activation or reduced osteoblastogenesis can disrupt bone homeostasis, eventually causing diseases such as osteoporosis, arthritis, and bone cancer [3,4]. Osteoporosis is a common bone disease that leads to an increased risk of fracture. The most common form of osteoporosis is caused by estrogen deficiency in menopausal females. Medications such as corticosteroids and anti-epileptics may also cause an imbalance between bone resorption and formation, which can result in osteoporosis [5].
Many anti-resorptive inhibitors including bisphosphonates, calcitonin, estrogen, and selective estrogen receptor modulators have been used to treat osteoporosis. These inhibitors maintain bone mass by inhibiting osteoclast function [6]. Estrogen replacement therapy is the most popular treatment to prevent and treat postmenopausal osteoporosis. However, long-term estrogen replacement therapy can increase the risk of endometrial and breast cancers. Therefore, many investigators have focused their efforts on developing a new anti-resorptive agent that does not have side effects [7–10]. Because osteoclasts function in bone resorption, specifically inhibiting osteoclasts has been considered the main target in numerous studies.
Osteoclasts are multinucleated giant cells formed by mononuclear progenitors of the monocyte/macrophage family via the sequential proliferation, differentiation, and fusion of hematopoietic precursor cells [11]. Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kB (RANK) ligand (RANKL) are essential factors for osteoclast differentiation [12]. In addition, several inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin (IL)-1b, contribute to osteoclastogenesis by modulating the induction of RANKL, osteoprotegerin, and M-CSF [7,13]. RANKL binding to the cell surface RANK receptor results in RANKL/RANK/TNFR associated factors (TRAF) complexes that sequentially activate NF- kB and mitogen-activated protein kinases (MAPKs), including c- Jun N-terminal kinase (JNK), p38 kinase, and extracellular signal-related kinase (ERK) [14]. This activation plays a key role in mediating osteoclast differentiation, activation, and survival. RANKL also activates the expression of transcription factors such as a nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and c-Fos, which are essential for osteoclast development [7,9]. Therefore, RANKL signaling is considered the main target of anti-resorptive agents that suppress osteoclast activation and bone loss.
Acteoside is the main active compound of Rehmannia glutinosa, which is used widely in traditional Oriental medicine [15,16]. Acteoside is a strong antioxidant and has anti-hepatotoxic, anti-inflammatory, and anti-nociceptive activities [17–20]. We previously found that acteoside decreases tyrosinase activity and melanin biosynthesis by regulating ERK signaling [21], protects against reactive oxygen species (ROS)-mediated gingival damage [22], and suppresses mycotoxin-mediated cell damage [23]. These effects are closely related to nucleosides' ability to remove ROS and regulate MAPK-mediated signaling. In particular, ROS are suggested to mediator RANKL-induced signaling pathways and cellular events in osteoclasts. Pretreatment with antioxidants inhibited RANKL-induced activation of NF-kB, ERK, and IkBa, thereby suppressing osteoclastogenesis [24]. These findings strongly suggested that, in addition to anti-inflammatory activity, antioxidant activity is crucial for an anti-resorptive agent, thus acteoside can suppress RANKL-induced osteoclastogenesis.
In the present study, we explored whether acteoside has a therapeutic effect on bone loss. We examined the effects of acteoside on osteoclast differentiation and bone resorption and the related cellular mechanisms using in vitro and in vivo experimental systems.
Cistanche acteoside suppresses RANKL-induced osteoclastogenesis
Materials and Methods
Ethics Statement
Animal care and use practices were approved by the Chonbuk National University Committee on Ethics in the Care and Use of Laboratory Animals (Permit No. CBU 2010-0007). All experiments in this study were carried out according to the guidelines of the Animal Care and Use Committee of the University.
Mice, Chemicals, and Laboratory Wares
Four-week-old female ICR mice were purchased from Orient Bio Inc. (Seoul, Korea) and housed at 2261uC and 5565% humidity on a 12 h light/dark cycle with free access to food and water. Acteoside (3,4-dihydroxy-bphenethyl-O-a-rhamnopyranosyl-(1R3)-4-O-caffeoyl-b-D-glucopyranoside; C29H36O15) (Fig. S1) was isolated from the leaves of Rehmannia glutinosa. Acteoside was dissolved in phosphate-buffered saline (PBS) before use. RANKL, TNF-a, IL-1b, IL-6, and M-CSF were purchased from R & D Systems (Minneapolis, MN, USA). Antibodies specific to c-Fos, p65, p-p65, p-ERK, ERK, JNK, p-JNK, NFATc1, IkBa, p-IkBa, and b-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for p-p38 and p38 were purchased from Cell Signaling Technology (Danvers, MA, USA). Calcium Assay and Osteocalcin (OC) EIA Kits were purchased from BioAssay Systems (Hayward, CA, USA) and Biomedical Technologies (Stoughton, MA, USA), respectively, to determine serum biochemical parameters. Mouse tartrate-resistant acid phosphatase (TRAP) Assay kit (Immunodiagnostic Systems, Scottsdale, AZ, USA) was also used to measure serum TRAP5b level. Unless otherwise specified, additional chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA), and laboratory wares were from SPL Life Sciences (Pochun, South Korea).
Cell Cultures
Bone marrow cells were obtained from the tibiae and femora of 6 week-old female ICR mice according to methods described previously [25]. The bone marrow suspension was incubated in a 100-mm culture dish in the presence of 50 ng/ml M-CSF. After 3 days, adherent cells were used as bone marrow macrophages (BMMs) to induce osteoclastic differentiation. Some bone marrow cells were also incubated for 48 h without M-CSF, and adherent cells were cultured in an osteoblast differentiating medium, as described elsewhere [25]. RAW264.7 macrophage cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L- glutamine, and antibiotics. These cells were used as a counterpart cell line for BMMs to explore the effect of acteoside on osteoclastogenesis.
Osteoclastic Differentiation and TRAP Staining
BMMs were pretreated with various concentrations (0–50 mM) of acteoside for 2 h before stimulating with 100 ng/ml RANKL. Culture media was replaced with fresh media on days 2 and 5. After 7 days of incubation, the cultures were fixed in 4% PBS- buffered paraformaldehyde and stained with TRAP using a Sigma Aldrich kit according to the manufacturer’s instructions. TRAP-positive cells were counted using optic microscopy, and cells containing 3 or more nuclei were considered to be osteoclasts. RAW 264.7 cells were also exposed to 100 ng/ml RANKL after pretreatment with acteoside for 2 h, and after 7 days of incubation, the cells were processed for TRAP staining.
Measurement of Cell Viability
Cell viability was determined using water-soluble tetrazolium salt (WST)-8 reagent. In brief, BMMs or RAW264.7 cells cultured in a growth medium containing 10% FBS and antibiotics were treated with 10 mM acteoside or a phenolic compound such as quercetin, luteolin, apigenin, or epigallocatechin-3-gallate (EGCG). WST-8 reagent was added into the cultures after 48 h of incubation. After incubating for an additional 4 h, the WST-8- specific absorbance was measured at 450 nm using a microplate reader (Packard Instrument Co., Downers Grove, IL, USA).
Bone Resorption Assay
BMMs (16105 cells/ml) were suspended in a-MEM containing 50 ng/ml M-CSF and 100 ng/ml RANKL, then divided across a 24-well plate coated with calcium phosphate nanocrystals (OAAS-24; Osteoclast Activity Assay Substrate, Oscotec Inc., Choongnam, South Korea) at a density of 26104 cells/cm2 with and without acteoside. After incubating for 7 days, the cells were removed from the plates with 5% sodium hypochlorite, and pit formation was observed under an optic microscope. The resorbed area was also measured by an image analyzer and expressed as a percentage of the control value.
Western Blot Analysis
Whole protein lysates were prepared in a lysis buffer as described elsewhere [26]. Cytosolic and nuclear proteins were prepared as described previously [25]. Equal amounts of protein extract were separated by 12–15% SDS-PAGE and blotted onto polyvinyl difluoride membranes. The blots were probed with primary antibodies overnight at 4uC before incubation with secondary antibody in blocking buffer for 1 h. The blots were
developed with enhanced chemiluminescence (Amersham Pharmacia Biotech Inc., Buckinghamshire, UK) and exposed to X-ray film (Eastman-Kodak Co., Rochester, NY, USA).
MAPK Activity Assay
Cells were pretreated with acteoside for 2 h and then stimulated with RANKL for an additional-30 min. MAPK activities were determined using immunometric assay kits, such as the p-p38 kinase assay kit (Assay Designs, Inc., MI, USA), p-ERK enzyme assay kit (Assay Designs), and p-SAPK/JNK sandwich ELISA kit (Cell Signaling Technology, MA, USA). All procedures followed the manufacturer’s instructions, and absorbance was measured by a microplate reader.
Electrophoretic Mobility Shift Assay (EMSA)
DNA-protein binding reactions were performed for 30 min at room temperature, with 10–15 mg protein in 20 ml buffer containing 1 mg/ml BSA, 0.5 mg/ml poly (dI-dC), 5% glycerol, 1 mM DTT, 1 mM PMSF, 10 mM Tris-Cl (pH 7.5), 50 mM NaCl, 30,000 cpm of [a-32P] dCTP-labeled oligonucleotides, and the Klenow fragment of DNA polymerase. The samples were separated on 6% polyacrylamide gels, which were dried and exposed to X-ray film (Eastman Kodak Co.) for 12–24 h at 270uC. The oligonucleotide primer sequences specific for NF-kB were 59-AAG GCC TGT GCT CCG GGA CTT TCC CTG GCC TGG A-39 and 39-GGA CAC GAG GCC CTG AAA GGG ACC GGA CCT GGA A-59.
NF-kB Luciferase Assay
RAW264.7 macrophages in 24-well plates were transfected with 0.8 mg kB-luciferase reporter vector using 2 ml of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. At 24 h after transfection, the cells were stimulated with RANKL in the presence and absence of acteoside for 24 h. Cells were resuspended in 100 ml reporter lysis buffer
(Promega, Madison, WI, USA). Equal amounts of protein samples were placed into 96-well microplates and mixed with luciferase substrate. Luminescence was measured by using a microplate luminometer (MicroLumat Plus LB 964, Berthold Technologies, Bad Wildbad, Germany). In this experiment, a permeable NF-kB inhibitor peptide (BIOMOL, Butler Pike, PA, USA) was used as a positive kB inhibitor.
Measurement of Cytokines
BMMs or RAW264.7 cells were stimulated with RANKL in the presence of acteoside in 24-well culture plates. After 48 h of incubation, culture supernatants were collected and assessed by ELISA using TNF-a-, IL-1b- and IL-6-specific OptEIATM kits according to the manufacturer’s instructions.
Real-Time Reverse transcription-polymerase Chain Reaction (RT-PCR)
The mRNA expression of osteoclastic markers, such as c-Fos, NFATc1, and TNF-a, was determined by real-time RT-PCR. In brief, total RNA was extracted from macrophages with Trizol reagent according to the manufacturer’s instructions (Invitrogen). cDNA was synthesized with 1 mg of total RNA using SuperScript Reverse Transcriptase II and primers (Invitrogen). Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA,USA) was used to detect the accumulation of PCR product during cycling with the ABI 7500 sequence detection system (Applied Biosystems). After denaturation at 95uC for 10 min, PCR was
performed using forty 3-step cycles of denaturation at 95uC for 15 sec, annealing at 60uC for 1 sec, and extension at 72uC for 30 sec. All PCR reactions were performed at least in triplicate, and the expression levels were normalized to the housekeeping gene HPRT in the same reaction. This study used the same primer sequences described elsewhere [7].
Measurement of Intracellular ROS
A stock solution of 29,79-dichlorodihydrofluorescein-diacetate (DCFH-DA) (50 mM; Calbiochem, Darmstadt, Germany) was prepared in DMSO and stored at 220uC in the dark. In brief, BMMs (106 cells/ml in 6-well plates) were cultured with 50 ng/ml M-CSF for 24 h and then treated with various concentrations (0– 10 mM) of acteoside 2 h before stimulation with 100 ng/ml RANKL. After 1 h of co-incubation, these cells were subsequently incubated with 25 mM DCFH-DA for 30 min. The green fluorescence of 29,79-dichlorofluorescein (DCF) was recorded at 515 nm (FL 1) using a FACS VantageH system (Becton-Dickinson, San Jose, CA, USA), and 10,000 events were counted per sample.
Induction of Ovariectomy-Induced Osteoporosis
Female ICR mice (6 week-old) were used for this study. Mice received a sham operation (Sham, n =10) or surgical ovariectomized (OVX, n=20) under anesthesia. One week after surgery, the OVX mice were randomly divided into 2 groups of 10 mice each: bilateral OVX and bilateral OVX supplemented with 200 ml
PBS containing 1 mM acteoside orally (AC group). Oral acteoside was administrated once every 3 days for 8 weeks after surgery, and
the same amount of PBS was administered to the Sham and OVX groups. After 1 day of the last administration, mice were sacrificed and then biochemical parameters in serum and 3-dimensional bone structure were analyzed.
Determination of Serum Biochemical Parameters
Blood samples were collected via cardiac puncture and serum was collected by centrifugation. Serum samples were stored at 280uC for analyses of biochemical parameters. The serum levels of IL-1b and IL-6 were estimated by using the ELISA kit as described above, while alkaline phosphatase (ALP) activity was determined by using a biochemical colorimetric assay that measures the amount of p-nitrophenol produced from a p-nitrophenol phosphate substrate, as described elsewhere [27]. To estimate the biomarkers of bone formation and resorption, serum OC, calcium, and TRAP5b levels were also determined according to the manufacturers’ instructions.
Analyses of Bone Structure and Morphometric Parameters
The femora of mice (Sham, OVX, and AC groups) were dissected and filled with physiological saline for mechanical testing. The mechanical strength of the femur was measured as described elsewhere [28]. The fracture load was recorded as the peak force in newtons at the point that the mid-shaft of the right femur fracture. In addition, the light femur of each animal was histo-morphometrically analyzed using a microcomputer tomography (micro-CT) system (SkyScan 1076 microfocus X-ray system, Kontich, Belgium). In brief, the bones in 4% formaldehyde storage were dried superficially on paper tissue before being wrapped in plastic ‘‘cling-film’’ or in parafilm, to prevent drying during scanning. Each plastic-wrapped bone was placed in plastic/polystyrene foam tubes which were mounted vertically horizontally in the 1076 scanner sample chamber for
micro-CT imaging. Scanning was carried out using 100 kV source voltage and 140 mA source current with 35 mm resolution.
Three-dimensional models of the trabecular bones of the femur were reconstructed using SkyScan CT Analyzer version 1.11. The structural parameters such as trabecular bone mineral density (BMD, g/cm3), percent bone volume (bone volume (BV)/tissue volume (TV), %), thickness (Tb.Th, mm), separation (Tb.Sp, mm), and number (Tb.N, 1/mm) were then measured.
Osteogenic Differentiation and Mineralization Assay Bone marrow cells cultured in 6-well culture plates were treated with DAG (10 nM dexamethasone, 50 mM ascorbic acid, and 20 mM b-glycerophosphate) in the presence of 10 mM acteoside. After 2 weeks of differentiation, the cells were fixed with ice-cold 70% (vol/vol) ethanol for 1 h and stained with 0.2% alizarin red S in distilled water for 30 min at room temperature. After the cells were destained and air-dried, the cell culture plates were evaluated by light microscopy using an inverted microscope (Nikon TS100, Japan). To quantify the amount of red dye, the stain was eluted with 10% acetyl pyridinium chloride by shaking for 20 min and the absorbance was measured at 560 nm. The amount of calcium deposited in the cell layers was also measured using a Calcium C kit (Wako Chemical Inc. Osaka, Japan) according to the manufacturer’s instructions. In addition, the expression of bone-specific mRNA markers, such as runt-related transcription factor-2 (Runx2), osterix, bone sialoprotein (BSP), and OC was determined by real-time RT-PCR. Oligonucleotide primers of these markers were designed with product sizes less than 200 bp using Primer Express Software 3.0 (Applied Biosystems) as described elsewhere [27].
cistanche acteoside boosts the bone formation
Results
Acteoside Inhibits Osteoclast Formation by Macrophages in a Dose-Dependent Manner
To verify the effect of acteoside on BMM differentiation into osteoclasts, the cells were cultured with various concentrations (0– 20 mM) of acteoside for 7 days in the presence of 50 ng/ml M- CSF and 100 ng/ml RANKL. Acteoside reduced the number of osteoclasts in a dose-dependent manner. When the cells were pretreated with 10 mM acteoside for 2 h, the osteoclast number decreased by 43% compared to cells supplemented with M-CSF and RANKL (Fig. 1A). Fig. 1B shows the RANKL-mediated osteoclast differentiation and its inhibition by combined treatment with acteoside. Consistent with this result, acteoside pretreatment decreased the RANKL-stimulated differentiation of RAW264.7 cells and osteoclast formation (Figs. 1C and D). When the anti- osteoclastic potential of acteoside on BMMs was compared with the antiosteoclast potentials of several phenolic compounds at the same concentration (10 mM), luteolin showed the highest activity (Fig. 2A). However, luteolin, quercetin, or apigenin itself decreased the viability of the cells (Fig. 2B). All the compounds inhibited osteoclastic differentiation by RAW264.7 macrophages with the following relative activities: luteolin. quercetin = apigenin .
Cistanche acteoside Inhibits Osteoclast
